Supplementary Information Contents SI 1- Archaeological context of newly reported individuals ................................ 2 SI 2- Experimental procedures ............................................................................. 63 SI 3- Y-chromosome analysis ................................................................................ 66 SI 4- f-statistics ....................................................................................................... 68 SI 5- qpGraph analysis ........................................................................................... 70 SI 6- qpAdm analysis .............................................................................................. 72 References ...................................................................................................................... 91 1 1 SI 1- Archaeological context of newly reported individuals 2 3 Dates 4 In what follows, we give dates in one of two formats. If there is no direct radiocarbon 5 date, we give a date based on archaeological context, in a format like “2500–1700 6 BCE”. If there is a direct radiocarbon date, we give a date in a format like “95.4% CI 7 calibrated radiocarbon age (Conventional Radiocarbon Age, Lab number),” using the 8 prefix “cal” to indicate a date that is obtained from skeletal material for the same 9 individual that yielded DNA (an example is “5983–5747 calBCE (6980±50 BP, Beta- 10 226472)”). We calibrated all dates in OxCal 4.31 using the IntCal13 calibration curve2. 11 We note that in the course of generating 62 new radiocarbon dates for this study, we 12 found that two individuals (RISE568 and RISE569) from Brandysek for which shotgun 13 sequencing data was originally reported in Allentoft et al. 20153 and labelled as being 14 from the Bell Beaker culture, were in fact misattributed. Our direct radiocarbon dating 15 supports a much later date for RISE569: 660-770 calCE (1300±30 BP, Poz-84461). 16 Thus, these individuals have not been included in our analysis. 17 Galeria da Cisterna (Almonda karst system, Torres Novas, Portugal) 18 Contact person: João Zilhão 19 Galeria da Cisterna is a fossil karst spring of the River Almonda, which now flows ~5 m 20 below ground at the base of a ~75 m high cliff. The length of this narrow, meandering 21 passage is approximately 100 m, and its cross-section is in general less than 2×2 m. The 22 current entrance was exposed in the 1920s by a landslide, which allowed access and 23 limited archaeological work carried out between the years 1937 and 19424–6. Three loci 24 were identified and focused on in subsequent excavations: AMD1, AMD2 and AMD3. 25 The AMD2 locus, excavated in the years 1988–89, featured a shallow Holocene deposit 26 containing funerary contexts with grave goods spanning the interval between the Early 27 Neolithic and the Iron Age. The lack of internal stratigraphic differentiation of this 28 Holocene deposit is primarily due to the repeated prehistoric and early historic human 29 habitation of the site, compounded by the activity of burrowing animals. A set of typical 30 Bell Beaker V-perforated ivory buttons and a small fragment of a gold spiral suggested 2 31 that a Bell Beaker component ought to exist among the human bone remains. This was 32 eventually corroborated by direct radiocarbon dating to this period of four right first 33 pedal phalanges7,8. Two produced genome-wide DNA: 34 I0839/AMD2-F23-90: 2457–2206 calBCE (3847±29 BP, OxA-28859) 35 I0840/AMD2-G18-187: 2456–2201 calBCE (3836±29 BP, OxA-28857) 36 Cova da Moura (Natural Cave, Torres Vedras, Portugal) 37 Contact person: Ana Maria Silva 38 The natural cave of Cova da Moura (Torres Vedras, Portugal) was discovered in 1930, 39 with excavations undertaken in 1932 and in 19619–11. This cave was used as collective 40 burial place between 3700 and 2200 BCE (Middle Neolithic to the Copper Age), 41 according to the results of seven radiocarbon dates obtained on human bones12,13. The 42 human remains were found commingled and fragmented. The study of the human 43 remains performed by Ana Maria Silva12,14 indicated a minimum number of 90 44 individuals, both sexes, and 15 non-adults. In terms of material 45 culture, Cova da Moura is by far the richest burial known in the region. Grave goods 46 include limestone and bone idols, green stone pendants, gold artefacts, engraved slate 47 plaques, bone, ivory, and variscite rabbit figurines, beads, and pre-Beaker and Beaker 48 ceramics9–11,15. Further studies of this bone assemblage include isotopic analysis for 49 dietary inferences16 and mobility17, as well as nonmetric dental traits to assess 50 populations affinities13. We generated genome-wide DNA data from one individual: 51 I4229/CDM3: 2289–2135 calBCE (3775±25 BP, PSUAMS-1750) 52 Paris Street (Cerdanyola del Vallès, Barcelona, Spain) 53 Contact person: Joan Francès Farré 54 During urban construction work at Paris Street in Cerdanyola del Vallès (Vallès 55 Occidental, Barcelona province) in 2003, a large amount of skeletal material and 56 associated pottery was unearthed. Follow-up excavation uncovered a Chalcolithic 57 hypogeum with more than 9,000 human remains as well as lithic and ceramic material, 58 the latter assigned to the Bell Beaker tradition18. 3 59 The hypogeum displays several occupational phases. The oldest one presented an ash 60 layer underlying the first inhumations that could have a ritualistic significance. Charcoal 61 from that basal layer was dated to 2878-2496 calBCE (4110±60 BP, UBAR-817). The 62 first funerary phase (UE-15) shows a large number of successive inhumations (minimal 63 number of individuals 36) that are still in anatomical position, placed in lateral 64 decubitus and with flexed knees. Seven arrow points were retrieved from this layer. A 65 thin, upper layer (UE-5) probably represents a re-organization of the existing funerary 66 space, prior to the second funerary phase (UE-2). At UE-5, two Bell Beaker vessels of 67 maritime style were retrieved. The UE-2 layer comprises fewer inhumations, and all of 68 them were accompanied by typical Bell Beaker vessels: three in Maritime style, and two 69 in epi-Maritime style. There were also numerous additional pieces of diverse typology. 70 Over this layer, a final one, labelled UE-3, contained two more skeletons arranged over 71 riverbed pebbles with a Bell Beaker vessel of a regional style known as "Pyrenaic". A 72 bone from this layer yielded the youngest date in the hypogeum of 2469-2206 calBCE 73 (3870±45 BP, UBAR-860). We recovered ancient DNA data from 10 individuals: 74 I0257/10362A: 2571–2350 calBCE (3965±29 BP, MAMS-25937) 75 I0258/10367A: 2850–2250 BCE 76 I0260/10370A: 2850–2250 BCE 77 I0261/10378A: 2850–2250 BCE 78 I0262/10381A: 2850–2250 BCE 79 I0263/10385A: 2850–2250 BCE 80 I0823/10360A: 2850–2250 BCE 81 I0825/10394A: 2474–2300 calBCE (3915±29 BP, MAMS-25939) 82 I0826/10400A: 2833–2480 calBCE (4051±28 BP, MAMS-25940) 83 I1553/10388A: 2850–2250 BCE 4 84 Arroyal I (Arroyal, Burgos, Spain) 85 Contact person: Manuel A. Rojo Guerra 86 The site of Arroyal I was excavated by a research team from the University of Burgos in 87 2011–2012. The site is a megalithic grave with well-preserved structural elements: a 88 rectangular chamber (3x3.5 m), a long corridor (6 m), and a stone mound. The grave 89 was used as a collective burial during 400 years in the Late Neolithic (3300–2900 90 calBCE)19. The grave was then abandoned until the Chalcolithic when it was 91 extensively remodelled: Neolithic layers were almost eliminated; the corridor was filled 92 with rocks and sediment; the useful area inside the chamber was reduced when a stone 93 wall was built; and a floor of limestone blocks was built inside the chamber. Several 94 consecutive and isolated burials (9–10) were then introduced. The last one (Roy5) was a 95 young individual buried with a set of 4 vessels (2 Bell Beakers and 2 carinated bowls) 96 and surrounded by the long bones and skulls from previous burials. She represents the 97 earliest observation of steppe-related genetic affinities in the Iberian Peninsula. Then 98 the dolmen was closed using materials from the site (in secondary position) and, at the 99 same time, the mound height was increased. Finally, an isolated pit grave (Roy4) was 100 made inside the mound. We successfully analysed 5 individuals from this site: 101 I0458/Roy1/SU25, Skull 1: 2458–2206 calBCE (3850±30 BP, UGA-15904) 102 I0459/Roy2/UE25, Isolated human jaw: 2600–2200 BCE 103 I0460/Roy3/SU25, Skull 2: 2461–2210 calBCE (3860±30 BP, UGA-15905) 104 I0461/Roy4/SU19, Inhumation 1: 2348–2200 calBCE (3827±25 BP, MAMS-14857) 105 I0462/Roy5/SU25, Inhumation 2: 2465–2211 calBCE (3870±30, UGA-15903); 106 2566–2346 calBCE (3950±26 BP, MAMS-25936) 107 Samples Roy1 and Roy3 were genetically first-degree relatives and belonged to 108 different mitochondrial haplogroups, which points to a father-son relationship. 109 Camino de las Yeseras (San Fernando de Henares, Madrid, Spain) 110 Contact person: Corina Liesau, Patricia Ríos, Concepción Blasco, Pilar Prieto 111 Most of our knowledge about this site has been gathered from four excavation 112 campaigns, three of which have been rescue archaeology interventions by different 5 113 companies. This has conditioned the information available about the site. The site of 114 Camino de las Yeseras is one of the greatest Chalcolithic ditched enclosures 115 (approximately 22ha.) in central Iberia. It is essential to our understanding of the 116 Chalcolithic period: the Pre Beaker burials and the impact that Bell Beaker customs and 117 funerary rituals had on the consolidation of social inequalities among the first 118 metallurgical societies of the Central Iberian Peninsula20,21. 119 Strategically located at the confluence of two important rivers, it was probably a central 120 place located on a suitable and well-communicated landscape comprising two valleys 121 for livestock and farming activities, and close to a rich resource catchment area where 122 flint, salt, and clay are found.